Due to recent advances in genetic and cell engineering technologies, proteins known to exhibit various pharmacological actions in vivo are capable of production in large amounts for pharmaceutical applications. Such proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interferons (alpha, beta, gamma, consensus), tumor necrosis factor binding protein (TNFbp), interleukin-1 receptor antagonist (IL-1ra), brain-derived neurotrophic factor (BDNF), kerantinocyte growth factor (KGF), stem cell factor (SCF), megakaryocyte growth differentiation factor (MGDF), osteoprotegerin (OPG), glial cell line derived neurotrophic factor (GDNF) and obesity protein (OB protein). OB protein may also be referred to herein as leptin.
Because these proteins generally have short in vivo half-lives and negligible oral bioavailability, they are typically administered by frequent injection, thus posing a significant physical burden on the patient and associated administrative costs. As such, there is currently a great deal of interest in developing and evaluating sustained-release formulations. Effective sustained-release formulations can provide a means of controlling blood levels of the active ingredient, and also provide greater efficacy, safety, patient convenience and patient compliance. Unfortunately, the instability of most proteins (e.g. denaturation and loss of bioactivity upon exposure to heat, organic solvents, etc.) has greatly limited the development and evaluation of sustained-release formulations.
Attempts to develop sustained-release formulations have included the use of a variety of biodegradable and non-biodegradable polymer (e.g. poly(lactide-co-glycolide)) microparticles containing the active ingredient (see e.g., Wise et al., Contraception, 8:227-234 (1973); and Hutchinson et al., Biochem. Soc. Trans., 13:520-523 (1985)), and a variety of techniques are known by which active agents, e.g. proteins, can be incorporated into polymeric microspheres (see e.g., U.S. Pat. No. 4,675,189 and references cited therein).
One such technique is spray-drying, wherein the polymer and active ingredient are mixed together in a solvent for the polymer, and then the solvent is evaporated by spraying the solution, leaving polymeric droplets containing the active ingredient. For a detailed review of spray drying see e.g. Masters, K., "Spray Drying Handbooks" (John Wiley & Sons, eds., New York 1984). Although the spray drying technique has proven useful in certain instances, it still suffers from the fact that biologically active proteins are often denatured due to contact with the organic polymer and solvent, or due to the heat generated during the spray drying processes.
Another technique which can be used to form microspheres is solvent evaporation. Solvent evaporation involves the dissolving of the polymer in an organic solvent which contains either dissolved or dispersed active ingredient. The polymer/active ingredient mixture is then added to an agitated continuous phase which is typically aqueous. Emulsifiers are included in the aqueous phase to stabilize the oil-in-water emulsion. The organic solvent is then evaporated over a period of several hours or more, thereby depositing the polymer around the core material. For a complete review of the solvent evaporation procedure see e.g. U.S. Pat. No. 4,389,330 (and references cited therein). As with the spray drying technique, solvent evaporation techniques have proven useful in certain instances. However, the technique is often not preferred because active ingredient is often lost during the solvent extraction process. This is because the process involves emulsification into an aqueous phase, and a water soluble drug will often rapidly partition from the more hydrophobic polymer-solution phase into the aqueous surroundings.
Yet another technique which can be used to form microspheres is phase separation, which involves the formation of a water-in-oil emulsion or oil in water emulsion. The polymer is precipitated from the continuous phase onto the active agent by a change in temperature, pH, ionic strength or the addition of precipitants. For a review of phase separation techniques, see e.g. U.S. Pat. No. 4,675,800 (and references cited therein). Again, this process suffers primarily from loss of active ingredient due to denaturation.
The release characteristics for the active ingredient from microparticles prepared by methods such as those described above may be continuous or discontinuous, and in some cases, the initial level of active ingredient release is too high or too low. Thus, various additives are often utilized in an attempt to control the release of active ingredient (see e.g., EP 0 761 211 A1, published Mar. 12, 1997).
To avoid the denaturation of protein and other fragile biological molecules which occurs upon spray drying, solvent evaporation or phase separation by classical techniques, the emulsion of polymers and active ingredient can be atomized into frozen nonsolvent overlayed with liquified gas such as nitrogen to form particles, and then extracted at very low temperatures. The extremely low processing temperatures may preserve the activity and integrity of the fragile biological molecules such as proteins. However, the method leads to poor loading efficiencies and yields, resulting in the loss of precious biological material, and is cumbersome, difficult and expensive to implement at the large scales required for commercial production.
Clearly the need still exists for an improved method for preparing polymeric microparticles containing an active ingredient which is simple, inexpensive, versatile, and, most importantly, which protects against loss of protein activity and which provides for high loading efficiencies and yields, thereby allowing for more consistent active ingredient release over an extended period of time.